Method for efficient production of biological particles comprising a viral or virus-like component in baffled rotating vessel

Abstract

The invention relates to a method of producing biological particles comprising a viral or virus-like component from producer cells, comprising the steps of: a) placing producer cells in a liquid medium in a vessel comprising a baffle structure inside the vessel; b) rotating the vessel so as to generate biological particles comprising a viral or virus-like component from the producer cells; and c) collecting the generated biological particles; wherein the step b) of rotating the vessel comprises repeatedly changing the rotational motion of the vessel.

Description

[0001] METHOD FOR EFFICIENT PRODUCTION OF BIOLOGICAL PARTICLES COMPRISING A VIRAL OR VIRUS-LIKE COMPONENT IN BAFFLED ROTATING VESSEL

[0002] TECHNICAL FIELD

[0003] The present invention relates to a method of producing biological particles comprising a virus or virus-like component for delivery of chemical and / or biological material, and to a vessel for performing the method.

[0004] TECHNICAL BACKGROUND

[0005] As the demand for gene and cell therapy vectors grows, scalable and cost- effective production of such vectors are increasingly important. The bioproduction of viruses or virus-like particles, which are often used as vectors for transporting and transferring both viral and no-viral genetic material, faces several challenges, particularly when scaling up for clinical and industrial applications.

[0006] The current gold standard for producing viruses in laboratory or clinical settings involves transfecting adherent mammalian cells in 2D static systems to produce viruses, for example lentiviruses, and after the culture, collecting a virusrich supernatant. This supernatant is then isolated and / or purified and concentrated to increase the virus titer.

[0007] However, this process has several challenges. First of all, producing viruses is cytotoxic to producer cells. Specifically, the viral fusion protein VSV-G, which is generally used to ensure the infectivity of the viral particles, leads to cell death of producer cells through syncytial formation. To address cytotoxicity, inducible competent stable cell lines (stable producer cell lines) can be engineered. However, creating these cell lines is time-consuming, often leads to lower production yield and may not be compatible with the testing of different therapeutic genes during preclinical stages. Therefore, transient transfection is often employed. However, this approach limits the operational timeframe of the producer cells (about 72h), i.e., until the cell death, multiplying the use of expensive materials and procedures and thus making large-scale production difficult. Moreover, the use of adherent mammalian cells has limited growth surface, impairing the scale-up and leading to a labor-intensive system that is not easy to automate. Current solutions to increase virus titer have thus focused on limiting the cytotoxic steps in the virus production and increasing cells surface. Strategies include using nanoparticles for transfection or growing producer cells in suspension. The growth of producer cells in suspension can reduce cytotoxicity by limiting cell-cell contact and the resulting syncytial formation, while increasing virus titer up to five-fold in some cases. Recent methods involve using suspension-grown producer cells where virus production is induced in the absence of antibiotics. The suspension cells are independent of the surface and can grow in bioreactors, which facilitates scaling-up production. Thus, it is more amenable to automation and closed systems, leading to low costs in virus production. The suspension techniques in serum-free media ease scaling but require adaptation of the producer cells.

[0008] For example, Tang, Q. L. et al., Bioengineered ; 12(1 ), 2095-2105 (2021 ) describes processes for functional lentivirus production in a stirred bioreactor using HEK293T cells in suspension.

[0009] Klimpel, M. et al., Biotechnology and Bioengineering; 120(9), 2622-2638 (2023) describes the use of suspension-adapted stable packaging cell lines for the development of a scalable and serum-free LV production process in bioreactors, in which the stable packaging cell lines are based on an inducible system.

[0010] Suleman, S. et al., Journal of Virological Methods; 325, 114884 (2024) describes lentiviral vector production from producer cells in suspension by identifying less toxic transfection reagents.

[0011] Overall, these suspension techniques do not seem easy to implement, are still not easily scalable given the high quantity of medium and materials required to process such as media, flasks, and animal serum, and require additional developments and optimization that may represent additional hurdles in a pre- clinical settings.

[0012] Production should also be versatile with respect to the configuration of the producer cells, including cells in suspension and in a 3D configuration, i.e., physiologically relevant, spheroid-like configuration. Several bioreactors for cell culture in the 3D configuration are known.

[0013] For example, document JP200722203 discloses a cell-culturing and centrifuging tube comprising a separation tube and a stopper for sealing the upper opening of the separation tube, an opening in the stopper, a culture gas- penetrable nonwoven fabric to cover the opening, and at least one baffle on the inner wall surface of the separation tube, which is integrally formed with the separation tube. There also exist commercially available systems for 3D cell culture, such as ClinoStar system and CERO 3D.

[0014] Some methods for producing extracellular vesicles, using a spinner flask bioreactor used for 3D cell culture, have also been developed.

[0015] For example, document FR3091296 discloses a fluidic system for loading a therapeutic or imaging agent into the lumen of extracellular vesicles from producer cells, comprising at least one vessel, a liquid medium contained by the vessel, producer cells, a liquid medium agitator and agitator speed control means adapted for the growth of the producer cells.

[0016] Document FR3091295 discloses a fluidic system for producing extracellular vesicles from suspended producer cells, comprising at least one vessel, a liquid medium contained by the vessel, suspended producer cells, a liquid medium agitator, agitator speed control means adapted for growth of the suspended producer cells.

[0017] Document FR3068361 discloses a fluidic system for producing extracellular vesicles from producer cells, including at least one container, a liquid medium contained by the container and producer cells, which also includes microcamers suspended in the liquid medium, the majority of producer cells being adherent to the surface of the microcarriers, and a liquid medium agitator, the agitator and the dimensions of the container being adapted to control a turbulent flow of the liquid medium in the container.

[0018] Document FR3112147 discloses a method for calibrating a fluidic system for producing extracellular vesicles from producer cells.

[0019] Document EP4015622A1 discloses a device for seeding cells which comprises a container with a wall, a bottom and a lid, wherein the container comprises a structured surface, and the structured surface is equipped to receive cells.

[0020] However, none of the above documents proposes using the bioreactors for production of viruses or virus-like particles. In any event, the devices and methods disclosed in the above documents pose several problems such as a low yield due to insufficient stress experienced by producer cells or the requirement of a large number of producer cells (e.g., 100 x 106cells).

[0021] Thus, there is a need for a more efficient and scalable method for producing biological particles comprising a viral or virus-like component, including viruses and non-infectious virus-like particles (VLPs) composed of viral proteins that selfassemble to mimic the structure of a native virus.

[0022] SUMMARY OF THE INVENTION The present invention relates to the following items.

[0023] Item 1 . A method of producing biological particles comprising a viral or virus-like component from producer cells, comprising the steps of: a) placing producer cells in a liquid medium in a vessel comprising a baffle structure fixed inside the vessel; b) rotating the vessel so as to generate biological particles comprising a viral or virus-like component from the producer cells; and c) collecting the generated biological particles comprising a viral or viruslike component; wherein the step b) of rotating the vessel comprises repeatedly changing the rotational motion of the vessel.

[0024] Item 2. The method of Item 1 , wherein the viral or virus-like component comprises a viral protein and / or a nucleic acid encoding the viral protein, the viral protein preferably comprising a structural protein such as a fusion protein, an envelope protein, a capsid protein, and other proteins involved in the virus structure, or a combination thereof.

[0025] Item 3. The method of Item 1 or 2, wherein the biological particles are selected from viruses or virus-like particles.

[0026] Item 4. The method of any one of Items 1 to 3, wherein the viruses are selected from retroviruses such as lentiviruses and Gamma-Retrovirus; adenoviruses; and Baboon virus, preferably lentiviruses.

[0027] Item 5. The method of any one of Items 1 to 4, wherein the biological particles are non-pathogenic, more preferably replication-deficient.

[0028] Item 6. The method of any one of Items 1 to 5, wherein the biological particles further comprises a non-viral molecule, preferably a therapeutic molecule, a biomarker, a bioactive protein or nucleic acid, an aptamer, an antibody and / or a molecular target.

[0029] Item 7. The method of any one of Item 6, further comprising, prior to step a), a step of introducing the non-viral molecule into the producer cells.

[0030] Item 8. The method of any one of Items 1 to 7, further comprising, prior to step a), a step of introducing at least one nucleic acid encoding the viral or virus-like component into the producer cells.

[0031] Item 9. The method of any one of Items 1 to 8, wherein the vessel rotates around a rotation axis which is substantially vertically oriented.

[0032] Item 10. The method of any one of Items 1 to 9, wherein the biological particles are generated from producer cells in the form of spheroids.

[0033] Item 11 . The method of Item 10, wherein: - the method further comprises a preliminary step of growing spheroids outside of the vessel, and step a) of placing the producer cells in the vessel comprises supplying the grown spheroids to the vessel; or

[0034] - step a) of placing the producer cells in the vessel comprises supplying individualized cells to the vessel, and the method further comprising an intermediate step of generating spheroids from the individualized cells.

[0035] Item 12. The method of any one of Items 1 to 11 , wherein the step b) of rotating the vessel comprises periodically changing the rotational motion of the vessel, preferably step b) comprises repeatedly reversing the rotational direction of the vessel, repeatedly changing the rotational speed of the vessel, and / or intermittently rotating the vessel.

[0036] Item 13. The method of Item 12, wherein the frequency of changing the rotational motion of the vessel is from 0.01 to 5 Hz, preferably from 0.05 to 0.5 Hz.

[0037] Item 14. The method of any one of Items 1 to 13, wherein the step b) of rotating the vessel is carried out at a maximum rotational speed of 50 to 6000 rpm, preferably 150 to 1200 rpm.

[0038] Item 15. The method of any one of Items 1 to 14, further comprising repeating cycles of at least steps b) and c), using the same producer cells.

[0039] Item 16. The method of any one of Items 1 to 15, wherein the baffle structure comprises one or more baffles fixed to an internal surface of the vessel.

[0040] Item 17. The method of any one of Items 1 to 16, wherein the vessel comprises a cylindrical inner wall and a central axis.

[0041] Item 18. The method of Item 17, wherein the baffle structure comprises one or more pairs of baffles, each pair of baffles comprising two diametrically opposed baffles relative to the central axis.

[0042] Item 19. The method of Item 17, wherein the baffle structure comprises a plurality of baffles extending from the cylindrical inner wall towards the central axis.

[0043] Item 20. The method of Item 19, wherein the baffles do not extend to the central axis.

[0044] Item 21. The method of Item 19, wherein the baffles extend to the central axis, thereby dividing the inside of the vessel into a plurality of compartments, the compartments being in fluid communication with one another.

[0045] Item 22. The method of Item 17, wherein the baffle structure comprises a plurality of baffles extending from the central axis towards the cylindrical inner wall. Item 23. The method of any one Items 17 to 22, wherein part or all of the baffles are solid plates.

[0046] Item 24. The method of Item 23, wherein the part or all of the plates are oriented substantially parallel to the central axis of the vessel.

[0047] Item 25. The method of Item 23 or 24, wherein part or all of the plates are oriented at an angle from 10° to 80°, preferably from 30° to 60° relative to the central axis of the vessel.

[0048] Item 26. The method of any one of items 1 to 25, wherein the vessel further comprises a coupling on an external surface of the vessel configured to be coupled to a rotating apparatus.

[0049] Item 27. The method of Item 26, wherein the coupling comprises one or more grooves or ridges on an external surface of the vessel.

[0050] Item 28. The method of Item 26 or 27, wherein the vessel further comprises a closing cap.

[0051] The present invention makes it possible to overcome the drawbacks of the prior art. In particular, the present invention provides an efficient method of producing biological particles comprising a viral or virus-like component from producer cells, and a vessel for performing the method.

[0052] This is achieved because the method comprises placing producer cells in a liquid medium in a vessel comprising a baffle structure inside the vessel, and rotating the vessel so as to generate biological particles comprising a viral or viruslike component from the producer cells, wherein the step of rotating the vessel comprises repeatedly changing the rotational motion of the vessel.

[0053] The step of rotating the vessel eliminates the need to use an impeller or an agitator. In this case, the vessel diameter determines the production volume, satisfying the need for miniaturization, which has been difficult to achieve with a conventional stirred tank with an impeller or an agitator, as explained above.

[0054] The present inventors have discovered that repeatedly changing the rotational motion of the vessel with a baffle structure is essential for the production of biological particles comprising a viral or virus-like component. Indeed, by repeatedly changing the rotational motion of the vessel with a baffle structure, the flow inside the rotating vessel is disrupted, resulting in a sufficient amount of shear stress to trigger an improved production of the biological particles comprising a viral or virus-like component in comparison with the conventional virus production as explained above.

[0055] In particular, the present invention provides the following advantages:

[0056] - a higher yield can be obtained compared with the production in suspension using a spinner flask bioreactor; - a low-volume regime (for example, a working volume in the range of 10 to 120 mL) is possible, which is perfectly in line with the number of cells per unit of volume associated with personalized medicine applications;

[0057] - ease of upscaling, with a volume of up to 10 L for massive production from producer cells;

[0058] - control of the flow conditions and thus the turbulent regime, is easier, allowing a more robust methodology in terms of the yield per cell;

[0059] - biological particles can be produced from cells in a 3D configuration, such as spheroids;

[0060] - biological particle production can be coupled with 3D cell culture or spheroid maturation.

[0061] BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 shows one example of a vessel of the invention.

[0063] Figure 2a shows a top view of an example of a vessel having a baffle structure comprising solid baffle plates of the invention.

[0064] Figure 2b shows a diagonal top view of the vessel shown in Figure 2a, cut along a plane parallel to the central axis of the vessel.

[0065] Figure 3a shows a top view of another example of a vessel with a baffle structure comprising solid baffle plates of the invention.

[0066] Figure 3b shows a diagonal top view of the vessel shown in Figure 3a, cut along a plane parallel to the central axis of the vessel.

[0067] Figure 4a shows the fluorescence intensity of the conditioned medium (CM) after virus-like particle (VLP) production in a vessel of the invention, as tested in Example 1 below. The y-axis represents relative fluorescence units (RFU) normalized by the number of producer cells. The x-axis represents the rotational speed of the vessel.

[0068] Figure 4b shows the number of VLPs produced per cell in a vessel of the invention, as tested in Example 1 below. The y-axis represents the number of released VLPs per producer cell. The values on the y-axis each represent values multiplied by 1 ,000. The x-axis represents the rotational speed of the vessel.

[0069] Figure 5a shows the fluorescence intensity of the CM after VLP production in a vessel of the invention, using, as producer cells, HEK293T spheroids formed in microwells, as tested in Example 2a below. The y-axis represents RFU normalized by the number of producer cells. The x-axis represents the rotational speed of the vessel. 1 indicates that the transfection was performed after the spheroid formation, and 2 indicates that the transfection was performed before the spheroid formation. Figure 5b shows the number of VLPs produced per cell in a vessel of the invention, using, as producer cells, HEK293T spheroids formed in microwells, as tested in Example 2a below. The y-axis represents the number of released VLPs per producer cell. The values on the y-axis each represent values multiplied by 1 ,000. The x-axis represents the rotational speed of the vessel. 1 indicates that the transfection was performed after the spheroid formation, and 2 indicates that the transfection was performed before the spheroid formation.

[0070] Figure 6a shows the fluorescence intensity of the CM after VLP production in a vessel of the invention, using, as producer cells, HEK293T spheroids formed in the vessel, as tested in Example 2b below. The y-axis represents RFU normalized by the number of producer cells. The x-axis represents the rotational speed of the vessel.

[0071] Figure 6b shows the number of VLPs produced per cell in a vessel of the invention, using, as producer cells, HEK293T spheroids formed in the vessel, as tested in Example 2b below. The y-axis represents the number of released VLPs per producer cell. The values on the y-axis each represent values multiplied by 1 ,000. The x-axis represents the rotational speed of the vessel.

[0072] Figure 7 shows NanoFCM (nano-flow cytometry) analysis of VLPs particles released from HEK293T spheroids formed in microwells (left) or formed in a rotating tube of the invention (right), as tested in Examples 2a and 2b. The y- axis represents the intensity of the green fluorescence signal detected on single particles, and the x-axis represents the side scatter signal, providing information about the particle’s size and granularity. “FITC-“ (signal below 100) indicates fluorescence-negative. “FITC+“ (signal above 100) indicates fluorescencepositive.

[0073] Figure 8 shows the fluorescence intensity of the CM after lentivirus production in a vessel of the invention, as tested in Example 3 below. The y-axis represents RFU normalized by the number of producer cells. The x-axis represents the rotational speed of the vessel. “CTL” refers to the control (conventional production of lentivirus). A corresponds to high acceleration (6000 rpm at 0.2 Hz) and B corresponds to low acceleration (100 to 300 rpm at 0.1 Hz).

[0074] Figure 9 shows the efficiency of transduction of HeLa cells with serial dilutions of lentiviruses produced according to the method of the invention, as tested in Example 4 below, with measurements taken 4 days after the transduction. The y-axis represents GFP-expressing cells (in %) (a) and the mean fluorescence intensity (MFI) (b). The x-axis represents the dilution factors (V / V) of the lentivirus supernatant. CTL, 0, 1 , 2, 3, 4, 5, 6 indicate a control, spheroids stimulated at 0 rpm, at 60 rpm, at 60 / 150 rpm, at 60 / 300 rpm, at 60 / 600 rpm, at 150 rpm, and at 300 rpm, respectively. “CTL” refers to the control (conventional production of lentivirus).

[0075] Figure 10 shows the efficiency of transduction of HeLa cells with serial dilutions of lentiviruses produced according to the method of the invention, as tested in Example 4 below, with measurements taken 7 days after the transduction. The y-axis represents GFP-expressing cells (in %) (a) and the MFI (b). The x-axis represents the dilution factors (V / V) of the lentivirus supernatant. CTL, 0, 1 , 2, 3, 4, 5, 6 indicate a control, spheroids stimulated at 0 rpm, at 60 rpm, at 60 / 150 rpm, at 60 / 300 rpm, at 60 / 600 rpm, at 150 rpm, and at 300 rpm, respectively. “CTL” refers to the control (conventional production of lentivirus).

[0076] Figure 11 shows the efficiency of transduction of Jurkat cells with serial dilutions of lentiviruses produced according to the method of the invention, as tested in Example 5 below, with measurements taken 7 days after the transduction. The y-axis represents GFP-expressing cells (in %) (a) and the MFI (b). The x-axis represents the dilution factors (V / V) of the lentivirus supernatant. CTL, 0, 1 , 2, 3, 4, 5, 6 indicate a control, spheroids stimulated at 0 rpm, at 60 rpm, at 60 / 150 rpm, at 60 / 300 rpm, at 60 / 600 rpm, at 150 rpm, at 300 rpm, respectively. “CTL” refers to the control (conventional production of lentivirus).

[0077] Figure 12a and Figure 12b show one example of a vessel subjected to orbital shaking (orbital shaker) which is not in accordance with the invention, as tested in Examples 6 to 9 below. Figure 12a shows a diagonal top view of the vessel, and Figure 12b shows a top view of the vessel shown in Figure 12a.

[0078] Figure 13 compares VLP production between the method of the invention and a method using an orbital shaker, from producer cells transfected with a plasmid encoding a fusion protein, as tested in Example 6 below. The y-axis represents the number of produced VLPs per cell (Figure 13a) and the fluorescence intensity of the CM after VLP production (Figure 13b). On the x-axis, NC, A, and B indicate a negative control (0 rpm in a non-adherent cell culture flask for 3h); the rotation in the vessel of the invention (600 rpm, 2 Hz, 3h); and the rotation in the orbital shaker (100 rpm, 3h), respectively. TO on the x-axis of Figure 13b indicates a baseline control (initial state at time 0).

[0079] Figure 14 compares VLP production between the method of the invention and a method using an orbital shaker, from producer cells transfected with a plasmid encoding a capsid protein, as tested in Example 7 below. The y-axis represents the number of produced VLPs per cell (Figure 14a), the fluorescence intensity of the CM after VLP production (Figure 14b), and the nucleic acid concentration in the CM after VLP production (Figure 14c). On the x-axis, “1” and “2” indicate a control for the effect of the plasmid (producer cells not transfected with any plasmids) and producer cells transfected with plasmids, respectively. NC, A, and B indicate a negative control (0 rpm in a non-adherent cell culture flask for 3h); the rotation in the vessel of the invention (600 rpm, 0.2 Hz, 3h); and the rotation in the orbital shaker (100 rpm), respectively. TO in Figure 14b indicates a baseline control (initial state at time 0).

[0080] Figure 15 compares lentivirus production using the vessel of the invention, an orbital shaker or a spinner flask, as tested in Example 8 below. The y-axis represents the average percentage of the fluorescent cells for each dilution of the lentivirus supernatant (Figure 15a) and the average fluorescence of the cells (Figure 15b). The x-axis represents the dilution factors (V / V) of the lentivirus supernatant. NC, A, B and C indicate a negative control (0 rpm in a non-adherent cell culture flask for 3h); the rotation in the vessel of the invention (600 rpm, 0.1 Hz); the rotation in the orbital shaker (100 rpm); and the rotation in the spinner flask (100 rpm), respectively.

[0081] Figure 16 shows the effect of different rotational motions on the lentivirus production using the vessel of the invention or an orbital shaker, as tested in Example 9 below. The y-axis represents the average percentage of the fluorescent cells for each dilution of the lentivirus supernatant (Figure 16a) and the average fluorescence of the cells (Figure 16b). The x-axis represents the dilution factors (V / V) of the lentivirus supernatant. NC, A1 , A2, A3 and B indicate a negative control (600 rpm and 0 Hz in the vessel of the invention); the rotation in accordance with the method of the invention (180 rpm, 2 Hz for A1 , 600 rpm, 0.1 Hz for A2, and 600 rpm, 0.2 Hz for A3, in the vessel of the invention); and the rotation in the orbital shaker (100 rpm), respectively.

[0082] DESCRIPTION OF EMBODIMENTS

[0083] The invention will now be described in more detail without limitation in the following description.

[0084] The term “biological particle” as used herein refers to a microscopic particle which originates from or is derived from living organisms or biological processes in the organisms.

[0085] The term “viral or virus-like component” as used herein refer to a part of a virus or a structure that mimics a part of a virus, such as viral proteins, nucleic acids, or other molecular structures.

[0086] The term “viral protein” as used herein refers to a protein that is encoded by a genetic material (DNA or RNA) present in a viral genome and / or a protein that has a similar function to a protein that is encoded by a genetic material (DNA or RNA) present in a viral genome. This means that a particle containing such a viral protein can mimic viral behavior, such as fusion with a receiving cell and / or delivering of a material. Viral proteins play various roles in the viral life cycle, such as infection and replication, and assembly of new viral particles.

[0087] The term “structural protein” as used herein refers to a viral protein that forms the physical structure or plays a major rule in the mechanical properties of the virus.

[0088] The term “envelope protein” as used herein refers to a protein that is present on (or embedded in) the surface of biological particles.

[0089] The term “fusion protein” as used herein refers to a specific type of envelope protein that facilitates the fusion of biological particles with the membrane of a host cell.

[0090] The term “capsid protein” as used herein refers to a protein that forms the outer shell, or capsid, of a virus. The capsid encloses and protects the viral genome, which can be either DNA or RNA, and plays a crucial role in the virus's ability to infect host cells.

[0091] The term “non-structural protein” as used herein refers to a viral protein that is involved in viral replication, virulence and immunomodulation in the host cell.

[0092] The term “virus” as used herein refers to a microscopic infectious agent which is modified or naturally occurring and may be used in various applications. A virus generally comprises a capsid and an envelope, and possibly viral genome (DNA or RNA).

[0093] The term “virus-like particle” as used herein refers to a molecular structure that contains at least one viral protein but is non-infectious because it contains no viral genetic material (DNA or RNA). It can be also referred to as a “virus-free particle”. In the present invention, a virus-like particle may be considered as a virus-free extracellular vesicle.

[0094] The term “cell” as used herein refers to the smallest fundamental structural and functional unit of living organisms, which can divide and multiply.

[0095] The term “producer cell” as used herein refer to a cell that is capable of secreting biological particles.

[0096] The term “spheroid” as used herein refers to a cellular structure consisting of more than one single cell, which has initially developed from a single or from multiple cells.

[0097] The terms “microcarrier” as used herein refers to a particulate matrix that allows the growth of producer cells adherent on its surface or within it. The matrix may be comprised of particles, preferably substantially spherical particles, having a maximum diameter of between 50 pm and 500 pm, and preferably between 100 pm and 300 pm. The microcamers are generally beads whose density is chosen to be substantially close to that of the liquid culture medium of the producer cells, thereby allowing the beads to remain suspended in the liquid culture medium by gentle mixing.

[0098] The term “vessel” as used herein refers to any type of container for containing liquid medium, such as a tube or a tank.

[0099] The term “vertical” or “vertically” as used herein refers to a direction which is perpendicular to the plane of the horizon and parallel to the direction of gravity.

[0100] Method for producing biological particles comprising a viral or virus-like component from producer cells

[0101] The present invention provides a method of producing biological particles comprising a viral or virus-like component from producer cells.

[0102] The method comprises: a) placing producer cells in a liquid medium in a vessel comprising a baffle structure inside the vessel, b) rotating the vessel so as to generate biological particles comprising a viral or virus-like component from the producer cells; and c) collecting the generated biological particles comprising a viral or viruslike component, wherein the step b) of rotating the vessel comprises repeatedly changing the rotational motion of the vessel.

[0103] The method of the invention is performed ex vivo.

[0104] The vessel will be as described in detail later. The vessel may be rotated owing to the rotating apparatus, which will be also described later.

[0105] By rotating the vessel, a flow of liquid medium within the vessel is achieved. Preferably, no addition of liquid medium and no withdrawal of liquid medium takes place during the rotation. Preferably, the vessel does not comprise a feeding line and / or a collecting line flu idical ly connected to the internal space of the vessel.

[0106] Preferably, the producer cells move freely under the effect of the flow of liquid medium. In other terms, preferably, the producer cells are comprised in the bulk of the liquid medium, and are not fixed to a wall of the vessel.

[0107] The rotation of the vessel is preferably such that the flow of liquid with the vessel is turbulent. Preferably, a turbulent regime characterized by a Reynolds number of greater than 1 ,000, greater than 3,500, preferably greater than 5,000 and most preferably greater than 6,000 or greater than 7,500 or greater than 10,000, may be obtained while the shear stress on cells inside the liquid can be kept minimal. At a point in time when there is a stationary regime, i.e. the average velocity of liquid in the vessel is equal to the velocity of the vessel, the global Reynolds number can be overall estimated as Re = R x V / v, wherein R is the internal radius of the vessel, V is the velocity of the vessel peripheral wall and v is the kinematic viscosity of the liquid. At a point in time when there is a transitional regime, i.e. a change in speed of rotation or a reversal of the rotational direction, the Reynolds number can be locally estimated as Re = Wx V' / v, wherein W is a characteristic dimension of a baffle element (such as the radial length of a baffle plate), V’ is the relative velocity between the liquid and the baffle element, and v is the kinematic viscosity of the liquid. Preferably, the global Reynolds number and / or a local Reynolds number as defined above is within one of the ranges cited above during at least part of the duration of step b).

[0108] The producer cells may move relative to the vessel at a velocity which can reach a maximum value of at least 25, at least 50, at least 75, at least 100, or at least 125 mm / s.

[0109] Preferably, there is no impeller in the vessel nor any other rotating or agitation element (such as a magnetic agitator), so that the flow of liquid is solely effected by the rotation of the vessel itself.

[0110] During step b), the vessel is preferably closed (such as by using the closure element described above). The vessel may be substantially filled with liquid medium during this step, i.e. may comprise no (or substantially no) gaseous headspace. In alternative variations, a gaseous headspace may be present. Preferably, the volume of gaseous headspace in the vessel, if present, may be less than 20%, or less than 10%, or less than 5%, or less than 2%, or less than 1 %, relative to the volume of liquid in the vessel.

[0111] In some embodiments, the viral or virus-like component may comprise a viral protein and / or a nucleic acid (e.g., DNA or RNA) encoding the viral protein.

[0112] In some embodiments, the viral protein may comprise a structural protein.

[0113] For example, the structural protein may comprise a fusion protein, an envelope protein, a capsid protein, a packaging protein, and other proteins involved in the virus structure, or a combination thereof, preferably an envelope protein.

[0114] In some embodiments, the viral protein may further comprise non-structural proteins.

[0115] For example, the non-structural proteins may comprise proteins responsible for replication, such as a protease, replicase, polymerase, or a transcription factor, proteins responsible for immunomodulation, regulatory and accessory proteins, and / or proteins responsible for efficient gene delivery.

[0116] Additionally or alternatively, as explained above, the viral protein may comprise a protein that has a similar function to that of any of the above structural and non-structural proteins (also can be referred to as a “virus-like protein”). Such a virus-like protein may be produced, for example, by way of a point mutation, domain swapping or gene editing technology.

[0117] In some embodiments, the viral or virus-like component may comprise a nucleic acid which encodes at least one of the above proteins.

[0118] In some embodiments, the biological particles may be non-pathogenic, preferably replication-deficient.

[0119] By “replication-deficient” is meant that, in some embodiments, the biological particles do not comprise, as a viral or a virus-like component, a non- structural protein, preferably a protein responsible for replication, or a nucleic acid encoding the non-structural protein, preferably the protein responsible for replication.

[0120] In some embodiments, the biological particles may be selected from viruses or virus-like particles.

[0121] For example, viruses may be selected from retroviruses such as lentiviruses and Gamma-Retrovirus; adenoviruses; and Baboon virus, preferably lentiviruses.

[0122] In some embodiments, the biological particles may be viruses, and the viruses may comprise at least a fusion protein and a capsid protein.

[0123] Additionally or alternatively, the biological particles may be virus-like particles, and the virus-like particles may comprise at least a fusion protein or a protein that has a similar function as the fusion protein.

[0124] In some embodiments, the biological particles may further comprise a non- viral molecule.

[0125] The non-viral molecule may be a therapeutic molecule, a biomarker, a bioactive protein or nucleic acid (e.g., DNA or RNA, preferably RNA) and / or a molecular target.

[0126] By “therapeutic molecule” is meant a substance designed to prevent, treat, or manage diseases and medical conditions.

[0127] Therapeutic molecules may include proteins, peptides, nucleic acids (e.g., DNA or RNA, preferably RNA), and / or small molecules having a low molecular weight of 1000 daltons or less.

[0128] By “bioactive protein or nucleic acid” is meant a protein or nucleic acid (e.g., DNA or RNA, preferably RNA) which can participate in adjusting biological activities of receiving cells, such as modulating the behavior of receiving cells and / or enhancing the action of therapeutic molecules.

[0129] By “molecular target” is meant a protein or nucleic acid which specifically binds to or interact with a particular target molecule or cell, such as an antibody targeting specific cells, e.g., T cells for specific gene (e.g., CAR gene) delivery, or an aptamer.

[0130] The nucleic acid may be DNA or RNA, preferably RNA selected from mRNA (messenger RNA), siRNA (small interfering RNA), miRNA (microRNA), sgRNA (single guide RNA). The RNA is preferably mRNA.

[0131] In preferred embodiments, the non-viral molecule is a nucleic acid, preferably RNA, more preferably mRNA.

[0132] In some embodiments, the method may further comprise, prior to step a), a step of introducing at least one nucleic acid (e.g., gene, preferably RNA, more preferably mRNA) encoding the viral or virus-like component into the producer cells.

[0133] In other embodiments, the method may further comprise, after step a) and prior to step b), a step of introducing at least one nucleic acid (e.g., gene, preferably RNA, more preferably mRNA) encoding the viral or virus-like component into the producer cells. In this case, the step of introducing at least one nucleic acid (e.g., gene, preferably RNA, more preferably mRNA) encoding the viral or virus-like component may be performed inside the vessel.

[0134] The step of introducing at least one nucleic acid (e.g., gene, preferably RNA, more preferably mRNA) encoding the viral or virus-like component into the producer cells may be performed using a conventional method.

[0135] For example, the step of introducing the nucleic acid encoding the viral or virus-like component into the producer cells may comprise introducing a plasmid containing at least one nucleic acid (e.g., gene, preferably RNA, more preferably mRNA) into the producer cells.

[0136] For example, in some embodiments, the biological particles may be viruses, and the method may further comprise a step of introducing one plasmid containing a gene encoding a fusion protein and one plasmid containing a gene encoding a capsid protein. Additionally or alternatively, the biological particles may be virus-like particles, and the method may further comprise a step of introducing one plasmid containing a gene encoding a structural protein, preferably a fusion protein.

[0137] The introduction of such a plasmid into the producer cells may be performed using a conventional method, for example by transfection, transduction, electroporation, and / or microinjection.

[0138] As other examples, the step of introducing the nucleic acid encoding the viral or virus-like component into the producer cells may comprise introducing the at least one gene by electroporation, chemical transfection, microinjection, gene editing techniques, and / or by way of viral vector. In some embodiments, the method may further comprise, prior to step a), a step of introducing at least one nucleic acid (e.g., gene, preferably RNA, more preferably mRNA) constituting or encoding a non-viral molecule into the producer cells.

[0139] In other embodiments, the method may further comprise, after step a) and prior to step b), a step of introducing at least one nucleic acid (e.g., gene, preferably RNA, more preferably mRNA) constituting or encoding a non-viral molecule into the producer cells. In this case, the step of introducing at least one nucleic acid (e.g., gene, preferably RNA, more preferably mRNA) constituting or encoding a non-viral molecule may be performed inside the vessel.

[0140] The non-viral molecule may be as described above.

[0141] In preferred embodiments, the non-viral molecule may be introduced with the viral or virus-like component.

[0142] For example, in some embodiments, the biological particles may be viruses, and the method may further comprise a step of introducing one plasmid containing a gene encoding a fusion protein, one plasmid containing a gene encoding a capsid protein, and one plasmid containing a nucleic acid constituting or encoding a non-viral molecule. Additionally or alternatively, the biological particles may be virus-like particles, and the method may further comprise a step of introducing one plasmid containing a gene encoding a structural protein, preferably a fusion protein, and one plasmid containing a nucleic acid constituting or encoding a non-viral molecule.

[0143] In some embodiments, the method may further comprise a step of introducing a non-viral molecule and / or an imaging agent into the liquid medium. In this case, the generated biological particles preferably comprise the non-viral molecule and / or an imaging agent.

[0144] The non-viral molecule may be as described above.

[0145] The imaging agent may be any substance that is used to enhance the visibility of specific organs, tissues, cells or physiological processes during medical imaging examinations.

[0146] The step of introducing a non-viral molecule and / or an imaging agent into the liquid medium may be performed before, during and / or after the steps a) to c).

[0147] In some embodiments, the producer cells may be selected from human cells, animal cells, and combinations thereof.

[0148] In some embodiments, the producer cells may be human cells, preferably healthy human cells. In some embodiments, the producer cells are not human embryonic derived cells and in particular are not human embryonic stem cells. Alternatively, the producer cells may be also pathological cells, for example cells derived from tissues and / or cancerous lines such as A673 cells or HeLa cells.

[0149] In some embodiments, the producer cells may be animal cells, preferably murine cells, for example murine MSC (murine mesenchymal stem cells) cells.

[0150] In some embodiments, the producer cells may be stem cells, in particular induced pluripotent stem cells, or multipotent cells. By way of example, the stem cells may be selected from multipotent mesenchymal cells, genetically modified cells, umbilical cord vein endothelial cells (HUVEC) or primary cells.

[0151] In other embodiments, the producer cells may be cell line cells, preferably an immortalized cell line, human monocyte line or human line of cells of hematopoietic origin derived from B lymphocytes, more preferably, HEK293 cells or their derivatives, THP-1 cells or Raji cells.

[0152] In some embodiments, the producer cells may be isogenic cells, i.e., they are derived from a subject, so that the biological particles produced by said producer cells, can then be administered to the subject or another subject (in order to prevent or treat a disease) or can otherwise be used ex vivo.

[0153] In some embodiments, the biological particles may be administered to a subject (in order to prevent or treat a disease) but the producer cells are not derived from this subject. In this case, the producer cells may be allogeneic cells, i.e. from the same species as the species of the said subject. Alternatively, the producer cells may be xenogeneic cells, i.e., from a species different from the species of said subject.

[0154] The subject is preferably human but can also be an animal.

[0155] The producer cells may be either adherent to a culture medium or nonadherent to a culture medium (also referred to as suspension cells).

[0156] In the case of adherent producer cells, the culture medium can be composed of microcamers which themselves are suspended in a liquid culture medium.

[0157] In some embodiments, the producer cells may be adherent producer cells detached from their culture medium and put in suspension, for example by a suitable treatment selected from an enzymatic treatment, a chemical treatment, a mechanical treatment or a combination thereof.

[0158] Preferably, the producer cells are in the form of individualized cells suspended in the liquid medium. By “individualized cells suspended in the liquid medium” is meant that the cells are separate from each other.

[0159] In some other embodiments, the producer cells may be in the form of cell aggregates. The term “cell aggregates” refers to an assembly of a plurality of producer cells that adhere to each other. Preferably, the producer cells are then in the form of spheroids.

[0160] In some embodiments, when single cells are considered as producers, the concentration of the producer cells in the liquid medium in the vessel when the biological particles are generated is from 10000 to 10 million cells per milliliter, preferably from 50000 to 2 million cells per milliliter, more preferably from 100000 to 1 million cells per milliliter, even more preferably from 200000 to 500000 cells per milliliter.

[0161] In some embodiments, when producer cells are cell spheroids, the spheroids have an average diameter of from 50 pm to 5 mm, preferably from 100 pm to 500 pm; the number of cells per aggregate is from 100 to 1 million, preferably from 1000 to 100000. The concentration of spheroids is from 50 to 20000 spheroids per milliliter, preferably from 100 to 5000 spheroids per milliliter, more preferably from 200 to 1000 spheroids per milliliter.

[0162] The liquid medium used in the invention for the production of biological particles may be a conventional liquid medium, such as FBS (fetal bovine serum), (serum-free) DMEM (Dulbecco's Modified Eagle Medium), RPMI (Roswell Park Memorial Institute) or serum-free media.

[0163] The vessel rotates around a rotation axis. It is noted that “rotating around a rotation axis" differs from orbital motion, such as a movement of a vessel, for example, on an orbital shaker, in which vessel follows in a circular trajectory without rotating around a rotation axis.

[0164] Most preferably, when the vessel has a central axis (which will be defined later), the rotation axis corresponds to the central axis of the vessel.

[0165] In some preferred embodiments, the rotation axis is substantially vertically oriented (in parallel to the direction of gravity).

[0166] In some embodiments, direct visualization of flow trajectories of the liquid within the vessel is carried out during the rotation. In such a configuration, it is possible to track the movement of (e.g. fluorescent) beads (or of cells or particles tied to such beads or labeled with fluorescent markers) in a plane of the rotating vessel, as it rotates, with an ultrafast camera. This can provide thorough analysis of the shear stress experienced by the cells during the rotation, resulting is a better control of the flow conditions.

[0167] In some embodiments, step b) of rotating the vessel is carried out at a maximum rotational speed of 50 to 6000 rpm, preferably 150 to 1200 rpm. In some embodiments, the maximum rotational speed may from 50 to 100 rpm; or from 100 to 200 rpm; or from 200 to 300 rpm; or from 300 to 600 rpm; or from 600 to 1000 rpm; or from 1000 to 1600 rpm; or from 1600 to 3000 rpm; or from 3000 to 6000 rpm. Step b) comprises repeatedly changing the rotational motion of the vessel. This means that the vessel does not rotate at a constant speed during the entirety of step b). The speed of rotation of the vessel changes multiple times during step b).

[0168] In some embodiments, step b) may comprise periodically changing the rotational motion of the vessel. This means that a certain pattern of rotational motion is repeated multiple times with a certain frequency.

[0169] The frequency of changing the rotational motion of the vessel may be from 0.01 to 5 Hz, preferably from 0.05 to 0.5 Hz.

[0170] For example, step b) may comprise repeatedly (e.g. periodically) reversing the rotational direction (from clockwise to counterclockwise and conversely).

[0171] Alternatively or additionally, step b) may comprise repeatedly (e.g. periodically) changing the rotational speed of the vessel.

[0172] Alternatively or additionally, step b) may comprise intermittently rotating the vessel. This means that there are resting sequences within step b) when the vessel does not rotate. Rotating sequences alternate with resting sequences. Successive rotating sequences may be characterized by the same rotational direction or by different rotational directions.

[0173] The duration step b) may be for example from 1 hour to 5 hours, e.g., approximately 1 , 2, 3, 4 or 5 hours.

[0174] In some embodiments, the biological particles may be generated from producer cells in the form of spheroids.

[0175] In some embodiments, the method may further comprise a preliminary step of growing spheroids outside of the vessel, and the step a) of placing the producer cells in the vessel comprises supplying the grown spheroids to the vessel.

[0176] In some embodiments, the above-described step of introducing the nucleic acid encoding the viral or virus-like component into the producer cells may be, if present, performed before the preliminary step.

[0177] In other embodiments, the above-described step of introducing the nucleic acid encoding the viral or virus-like component into the producer cells may be, if present, performed after the preliminary step. In other words, the nucleic acid encoding the viral or virus-like component may be introduced into the producer cells in the form of spheroids.

[0178] The preliminary step of growing spheroids outside of the vessel is well known in the domain, for example, using hanging drop methods, microwell-based methods, scaffold-based methods, or agitation-based methods.

[0179] In other embodiments, the step a) of placing the producer cells in the vessel may comprise supplying individualized cells to the vessel, and the method may further comprise an intermediate step of generating spheroids from the individualized cells (before step b)).

[0180] The intermediate step may comprise rotating the vessel during the intermediate step of generating spheroids from the individualized cells, preferably at a maximum speed of rotation which is less than a maximum speed of rotation during step b).

[0181] In some embodiments, the above-described step of introducing the nucleic acid encoding the viral or virus-like component into the producer cells may be, if present, performed before supplying individualized cells to the vessel.

[0182] The rotational speed during the intermediate step may be from 20 to 200 rpm, preferably from 50 to 100 rpm. During this intermediate step, the rotation motion may remain constant. Alternatively, repeated changes of rotational motion may take place, as describe above in connection with step b).

[0183] In some embodiments, step c) of collecting the generated biological particles may be carried out by withdrawing the liquid medium including the producer cells from the vessel, and separating the biological particles from the withdrawn liquid medium.

[0184] The produced biological particles may be separated from the withdrawn liquid medium by conventional methods, such as by centrifugation, filtration, sizeexclusion chromatography, immunoaffinity-based separation, decantation, and any combination thereof.

[0185] In other embodiments, step c) may be carried by withdrawing the liquid medium from the vessel without substantially withdrawing the producer cells, and separating the biological particles from the withdrawn liquid medium, by any separation method as described above, preferably centrifugation.

[0186] In this case, decantation and / or centrifugation in the vessel itself is preferably carried out prior to withdrawing the liquid medium.

[0187] In some embodiments, the method may further comprise repeating cycles of at least step b) of rotating the vessel and step c) of collecting the generated biological particles, using the same producer cells.

[0188] For example, at each cycle, the producer cells may be withdrawn from the vessel; separated from the liquid medium (by way of, for example, centrifugation); and placed again in the vessel together with fresh liquid medium.

[0189] Alternatively, at each cycle, the liquid medium may be withdrawn from the vessel, the producer cells substantially remaining within the vessel (by way of, for example, centrifugation), and fresh liquid medium may be added to the vessel.

[0190] The method may further comprise a time interval of rest between two successive cycles. The producer cells may be kept in the vessel without rotation of the vessel during the time interval of rest.

[0191] The producer cells may be also kept in a conventional cell culture apparatus, such as a cell culture flask, without rotation during the time interval of the rest.

[0192] The producer cells may be also kept in the vessel in rotation (this may enable cell growth during the time interval of the rest). If the vessel rotates during rest, the rotation may be constant. The rotational speed (if constant) or the maximum rotational speed (if not constant) is less than the rotational speed during the cycles of step b). The rotational speed during any rest step may be from 20 to 200 rpm, preferably from 50 to 150 rpm.

[0193] For example, the method may comprises 2, 3, 4, 5, 6, 7, 8, 9 or 10 cycles. The duration of each cycle (optionally step a) and steps b) and c)) may be from 1 hour to 5 hours, e.g., approximately 1 , 2, 3, 4 or 5 hours. The duration of the time interval of rest between successive cycles may be from 30 min to 24 hours, for example, approximately 30 min, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 10 hours, 15 hours, 20 hours, 24 hours.

[0194] Application of the produced biological particles

[0195] The present invention may also relate to the use of biological particles for therapeutic purposes and / or for diagnostic purposes, such as personalized medicine, immunotherapy, regenerative medicine, cell therapy, and / or the treatment of tumors, infectious diseases, inflammatory diseases, immunological diseases, metabolic diseases, cancerous diseases, genetic diseases, degenerative diseases or diseases secondary to surgery or trauma.

[0196] For such purposes, the biological particles produced according to the present invention may be used as a vector or a carrier for delivering at least one therapeutic molecule, a biomarker, bioactive protein or nucleic acid (e.g., DNA or RNA, preferably RNA) and / or a molecular target, by way of administration to a subject in need thereof.

[0197] In some embodiments, the biological particles according to the invention may be used to modify immune cells, such as monocytes, THP-1 or lymphocytes, and may be used, for example, in immunotherapy and / or cancer therapy.

[0198] For example, the biological particles according to the invention may be used as a vector for delivering a CAR gene (Chimeric Antigen Receptors) to immune cells, preferably T cells. CAR (Chimeric antigen receptor) is a synthetic construct that is expressed in T cells to mimic the T cell activation and to target them towards a specific antigen. Thus, the biological particles according to the invention may be used to modify T cells to generate CAR-T cells, which may be used in immunotherapy and / or cancer therapy.

[0199] In other embodiments, the biological particles may be used to modify mesenchymal stem cells (MSC), and may be used in regenerative medicine for processes such as angiogenesis or wound healing.

[0200] In other embodiments, the biological particles can be used for vaccination, in the treatment of inflammatory diseases owing to their potential for antiinflammatory effects, or in the treatment of tumors, infectious diseases, immunological diseases, metabolic diseases, cancer diseases, genetic diseases, degenerative diseases or diseases secondary to surgeries or trauma.

[0201] Vessel for producing biological particles from producer cells

[0202] The present invention may be performed in a vessel for producing biological particles from producer cells. The vessel has an internal space for holding liguid medium. The vessel comprises a baffle structure inside the vessel, i.e. within the internal space. Preferably, the vessel comprises a coupling on an external surface configured to be coupled to a rotating apparatus as will be described in more detail below.

[0203] By “baffle structure” is meant one or more elements which are fixed within the vessel and which deviate the flow of liguid within the vessel. The baffle structure generally promotes turbulent flow in the vessel.

[0204] Fig. 1 shows one example of a vessel of the invention.

[0205] The vessel 1 comprises a baffle structure inside the vessel (not shown in Fig. 1), which will be explained in detail later, and a coupling 3 on an external surface of the vessel configured to be coupled to a rotating apparatus.

[0206] The vessel may be made of a suitable material for the production of biological particles, e.g., a biocompatible resin, a biocompatible polymer, or a metal.

[0207] The vessel may be manufactured by 3D printing, injection molding, blow molding, or compression molding, preferably by 3D printing and injection molding.

[0208] In some embodiments, the vessel has not been subjected to (or has not undergone) any surface treatment.

[0209] Preferably, and as illustrated, the internal space of the vessel is structurally delimited by a base and a peripheral wall extending from the base. The base is preferably substantially planar (flat). Preferably, the baffle structure does not cover the entirety of the peripheral wall. Preferably, the peripheral wall is smooth, i.e. does not include wells. Preferably, the shape of the vessel is substantially cylindrical (or at least the shape of the internal space of the vessel is substantially cylindrical). In this case, the peripheral wall is a cylindrical inner wall, as shown on Fig. 1. The central axis of the vessel can then be defined as the axis of the cylinder. Preferably, the axis is perpendicular to the base and the cylinder is a right cylinder. Preferably, the cylinder is a circular cylinder, more preferably a right circular cylinder. In alternative embodiments, the peripheral wall may be in the shape of a non-circular cylinder. For example, if the base is substantially in the shape of a polygon (such as a square or a rectangle), the peripheral wall may be in the shape of a cylinder composed of a number of planar sections joined along respective edges thereof (such as four planar sections).

[0210] The capacity volume of the vessel may be suitably adjusted depending on, for example, the working volume, and the target number of the biological particles to be produced.

[0211] In some embodiments, the vessel may have a capacity of from 10 mL to 10 L. For example, the capacity of the may be from 10 mL to 5 L, from 10 mL to 1 L, from 10 mL to 500 mL, or from 10 mL to 250 mL.

[0212] The internal diameter of the vessel may be suitable adjusted to accommodate different working volumes. For example, the internal diameter of the vessel may be from 2 to 25 cm. For example, the internal diameter of the vessel may be from 2 to 20 cm, from 3 to 15 cm, or from 3 to 10 cm.

[0213] The height (maximal dimension in the direction parallel to the central axis) of the vessel may be from 1 to 30 cm, preferably from 2 to 20 cm, and more preferably from 3 to 10 cm.

[0214] The vessel may be provided with a closure element, such as a cap. In this case, the vessel may comprise a neck at the top part of the vessel (opposite the base at the bottom part), which is designed to receive the cap to seal the vessel.

[0215] The size and shape of the neck may vary depending on the type of vessel and the cap. Generally, the neck has a smaller diameter than the rest of the vessel (also referred to as “body” of the vessel), having a shoulder (transition between the body and the neck), which may be a curved or sloping part of the vessel where the diameter changes from the body to the neck. The cap may be fixed to the vessel by a threaded engagement, by a bayonet connection, by friction fitting, by a magnetic connection or the like.

[0216] The coupling may have any suitable geometry, provided that the geometry allows the vessel to be rotatably fixed to the rotating apparatus.

[0217] In some embodiments, the coupling may comprise one or more grooves or ridges on the external surface of the vessel. For example, as shown in Fig. 1 , the coupling 3 may comprise a ridge (a raised or protruding line) running along the external surface of the vessel in the direction parallel to the central axis of the vessel (on the surface across from the peripheral wall in the internal space). The same is possible with a groove running along the external surface of the vessel in the direction parallel to the central axis of the vessel.

[0218] Alternatively or additionally, the coupling 3 may be provided on a bottom external surface of the vessel (across from the base in the internal space).

[0219] The baffle structure may extend from the peripheral wall of the vessel, towards the central axis of the vessel.

[0220] In some embodiments, the baffle structure may comprise one or more baffles. By “baffle" is meant a wall, which can be either substantially planar or curved, and preferably is substantially planar. The baffles are fixed in the internal space of the vessel, to an internal surface of the vessel. Preferably, they are fixed to the base and / or to the peripheral wall and are preferably integrally formed with the base and / or the peripheral wall. Each baffle acts as an obstacle to the flow of liquid within the vessel and is configured to deflect such flow of liquid. In the present text, when A is said to be “fixed to” B, it may mean “indirectly fixed to” ( / .e. A is fixed to B via an intermediate element); or, more preferably, “directly fixed to” ( / .e. A is fixed to B without any intermediate element between A and B).

[0221] For example, the baffle structure may comprise one baffle, two baffles, three baffles, four baffles, five baffles, or six baffles, seven baffles, eight baffles, nine baffles or ten baffles which is / are fixed to an internal surface of the vessel.

[0222] When the baffle structure comprises two or more baffles, the baffles may be regularly spaced within the vessel or may be non-regularly spaced within the vessel.

[0223] In some embodiments, the baffles may be fixed on the internal surface of the vessel symmetrically relative to the central axis of the vessel.

[0224] The baffles may have either the same dimensions (e.g., height, thickness, length), or different dimensions from each other.

[0225] For example, the height of each baffle may be from 1 to 25 cm, preferably from 1 to 15 cm, and more preferably from 1.5 to 6 cm. The term “height” for a baffle as used herein refers to the maximum dimension of the baffle in the direction parallel to the central axis.

[0226] The length of each baffle may be from 1 to 12.5 cm, preferably from 1 to 5 cm, and more preferably from 1 to 2.5 cm. The term “length” for a baffle as used herein refers to the maximum dimension of the baffle perpendicular to the central axis (and preferably parallel to the base of the vessel). The thickness of each baffle may be from 0.1 to 5 cm, preferably from 0.1 to 2.5 cm, and more preferably from 0.1 to 0.5 cm. The term “thickness” for a baffle as used herein refers to the dimension which is smaller than the maximum dimensions in the orthogonal directions and which is perpendicular to the length direction (and preferably parallel to the base of the vessel).

[0227] In some embodiments, the baffle structure comprises one or more pairs of baffles. Each pair of baffles may comprise two diametrically opposed baffles relative to the central axis of the vessel.

[0228] Preferably, the two baffles of each pair are identical in terms of dimensions.

[0229] For example, the baffle structure may comprise one pair, two pairs, three pairs, four pairs, or five pairs of the diametrically opposed baffles.

[0230] When the baffle structure comprises two or more pairs of baffles (for example, n pairs), the pairs may be arranged such that the baffles (for example, 2n baffles) are spaced apart equidistantly from each other. Alternatively, the pairs may be arranged such that the baffles (for example, 2n baffles) are spaced apart non-equidistantly from each other.

[0231] In some embodiments, the pairs of baffles may be symmetrically arranged relative to the central axis of the vessel.

[0232] The two baffles in each pair may have either the same dimensions among all pairs, or different dimensions among pairs.

[0233] In some embodiments, the baffle structure may comprise a plurality of baffles extending from the peripheral wall towards the central axis.

[0234] The baffles may be directly joined together. For example, the baffles may extend to the central axis and be joined at the central axis. In this case, the baffle structure may divide the internal space of the vessel into a plurality of compartments, which are in fluid communication with one another.

[0235] Alternatively, the baffles are not directly joined together (but are only indirectly joined via the base or the peripheral wall). In particular, the baffles may not extend up to the central axis, thus leaving an unobstructed central space in the vessel. In this case, the baffles may extend a different distance towards the central axis, or the baffles may extend the same distance towards the central axis.

[0236] In other embodiments, the baffle structure may comprise a plurality of baffles extending from the central axis of the vessel towards the peripheral wall of the vessel.

[0237] In some embodiments, the baffles do not extend to the peripheral wall of the vessel, thus leaving an unobstructed peripheral space between the baffles and the peripheral wall. Part or all of the baffles may be plates or walls, i.e. substantially flat elements. These elements may have one dimension, namely the thickness, which is much smaller (such as at least 10 times or 100 times smaller) than the maximum dimensions in the orthogonal directions.

[0238] The plates or walls are preferably substantially planar but may alternatively be curved.

[0239] When a plate or wall is substantially planar, the thickness is the dimension of the plate perpendicular to the main plane of the plate or wall. The thickness may vary or be constant across the structure. If it varies, then any thickness values are meant to designate the average thickness.

[0240] The plates or walls may be solid plates or walls.

[0241] The plates or walls may have an outer shape (perpendicular to the direction of the thickness) which may be substantially square, rectangular, triangular, trapezoidal, diamond, pentagonal, hexagonal, octagonal, more generally polygonal, or which may be at least partly curved. The plates or walls may in particular be substantially perpendicular to the base and may be oriented substantially parallel to the central axis of the vessel.

[0242] Alternatively, the plates or walls may be oriented substantially perpendicular to the central axis of the vessel.

[0243] The plates or walls may be also be oriented at an angle from 10° to 80°, preferably from 30° to 60° relative to the central axis of the vessel, and / or relative to the base.

[0244] For example, the plates or walls may be oriented at an angle of approximately 10°, 20°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 70°, or 80° relative to the central axis of the vessel and / or relative to the base.

[0245] All angle values in the present text are absolute values.

[0246] All baffles may be oriented similarly or not.

[0247] Below, several possible designs of the vessel of the invention will be explained more in detail by reference to the drawings, but it is understood that the design of the vessel can be configured and further modified to achieve optimal conditions flow conditions within the vessel.

[0248] Making reference to Fig. 2a and Fig. 2b, the vessel 1 may be substantially cylindrical in shape and may comprise a baffle structure 2 inside the vessel and a coupling 3.

[0249] The vessel may further comprise a neck T at the top part of the vessel and a body 1”. The body 1” comprises the base 7 and peripheral wall 8 as described above in connection with Fig. 1 The baffle structure 2 may comprise three pairs of baffles 4, each pair of baffles comprising two diametrically opposed baffles relative to the central axis of the vessel. A different number of pairs of baffles is of course possible.

[0250] In this example, the baffle structure 2 comprises six baffles 4 in total fixed to an internal surface of the vessel (the peripheral wall 8 and / or the base 7), and the three pairs are arranged such that the six baffles are regularly spaced apart along the circumference of the cylinder.

[0251] All of the baffles may be solid plates as shown, but the baffles may be also meshed or perforated plates, or a combination of solid plates, meshed plates and perforated plates.

[0252] The plates may have, but not limited to, a rectangular shape.

[0253] The baffles may extend from the peripheral wall 8 towards the central axis, without extending to the central axis (in other words, the baffles do not reach all the way to the central axis of the vessel and leave an unobstructed central space).

[0254] The two baffles 4 in each pair may be identical within the pair, but the dimensions may differ from pair to pair. For example, the baffles may be plates having the same thickness and the same length, but may differ in height among the pairs. The term “height” as used herein (for a plate, baffle or other structure) refers to the maximum dimension of the plate, baffle or other structure in the direction parallel to the central axis.

[0255] The term “length” as used herein (for plate, baffle or other structure) refers to the maximum dimension of the plate, baffle or other structure perpendicular to the central axis (and preferably parallel to the base 7).

[0256] Alternatively, the two baffles 4 in each pair may have, among the pairs, the same height, length, and thickness (identical among pairs); or the same thickness but different heights and lengths; or the same height and thickness but a different length; or the same height and length but a different thickness; or the same height but different thickness and length; or the same length but different height and thickness; or different height, length, and thickness.

[0257] The planes of part or all of the plates may be oriented substantially parallel to the central axis of the vessel.

[0258] For example, as shown in Fig. 2a and 2b, the planes of all of the plates may be oriented substantially parallel to the central axis of the vessel.

[0259] As a general remark, in all embodiments described therein, rounding or fillets may be provided at each edge or corner in order to avoid sharp edges which may induce local high shear areas which may damage any biological material present in the vessel. In the example of Fig. 2a and 2b, the edges of the baffles 4 facing towards the central axis and preferably running parallel to the central axis are rounded.

[0260] The vessel shown in Fig. 3a and Fig. 3b is the same as the vessel of the example shown in Fig. 2a and Fig. 2b, except that there is in addition a fillet in the area where the baffles 4 are connected to the peripheral wall 8 and to the base 7, allowing for a smoother transition from each baffle to the peripheral wall 8 and the base 7.

[0261] The arrangement, the orientation, and the dimension of the baffles may be as defined above.

[0262] System for producing biological particles from producer cells

[0263] The present invention may be also performed in a system for producing biological particles from producer cells.

[0264] The system comprises a vessel as defined above, and a rotating apparatus.

[0265] The vessel may be configured to be rotationally fixed to the rotating apparatus by keying the coupling of the vessel to a corresponding coupling on the rotating apparatus. If the coupling of the vessel comprises grooves, the coupling on the rotating apparatus may comprise corresponding ridges. If the coupling of the vessel comprises ridges, the coupling on the rotating apparatus may comprise corresponding grooves.

[0266] In some embodiments, the rotating apparatus may comprise a rotating element such as a cup configured for receiving the vessel. The coupling on the rotating apparatus may be present on an internal surface of the cup which is in contact with an external surface of the vessel.

[0267] Alternatively or in combination, the rotating apparatus may comprise a securing mechanism for maintaining the vessel rotatably fixed within the rotating element, comprising for example tightening means using screws, or a friction fit engagement.

[0268] In some embodiments, the outer shape of the vessel is non circular and the cup has a corresponding shape, thus ensuring that the vessel is rotationally fixed relative to the cup without any requirement for respective couplings on the external surface of the vessel and on the internal surface of the cup. For example, the vessel may comprise one or more planar external surfaces in addition to the base (e.g. the vessel may have an overall cuboid shape), or may comprise an ellipsoidal surface. In this case, the cup has a complementary shape so as to ensure contact between one or more external surfaces of the vessel and one or more internal surfaces of the cup. In some embodiments, the rotating apparatus may comprise a drive mechanism and a control unit for controlling the drive mechanism.

[0269] The rotating element (e.g. cup) may be actuated by the drive mechanism. The rotating apparatus may comprise a stationary part which may include the drive mechanism and the control unit.

[0270] Preferably, the control unit may be configured for implementing the rotation of the vessel as required by the method of the invention (which will be explained below).

[0271] The control unit may comprise one or more processors coupled to a storage medium, as well as a computer program comprising instructions stored thereon, for performing the various steps described in more detail below. The control unit may receive input from sensors in or associated with the rotating apparatus and / or input from the user. The control unit may process the input data and, as a result, provide instructions to the drive mechanism. In some embodiments, part or all of the control unit may be provided not in the rotating apparatus itself but in a separate computing device.

[0272] EXAMPLES

[0273] Preparation of plasmids

[0274] The packaging plasmid psPAX2 (12260; Addgene) and envelop plasmid pVSVG (pMD2.G; 12259, Addgene) were obtained from Addgene. The plasmid containing the gene of GFP (pWxId) was obtained by cloning of PCR-amplified GFP-2A-Puro sequence using the restriction enzyme BsrGI / Xhol (NEB). All the plasmids were used for heat shock transformation of competent cells DH5alpha (NEB) and culture in 250 mL of Luria Broth media overnight 37 °C and 200 rpm. The culture of bacteria was centrifuged at 3500 rpm, 15 min RT and the plasmids were purified using the NucleoBond Xtra Midi kit (Macherey Nagel) according to manufacturer’s instructions.

[0275] Spheroid formation in a vessel of the invention (Examples 2 to 5)

[0276] HEK293T cells were detached with trypsin, counted, and resuspended at a density of 1 million cells per mL. 20 mL of the cells suspension were added to the vessels of the invention (also referred to as “baffled tubes”), that were rotated for 48 hours, at 60 rpm, for 4 s clockwise, then a pause of 2 s, and then 4 s counterclockwise; collected spheroids were 150-300 pm in diameter.

[0277] Small spheroid formation in a vessel of the invention (Examples 6 to 9) To maximize direct transfection of spheroids with plasmids, smaller spheroids were produced. Specifically, HEK293T cells were detached with trypsin, counted, and resuspended at a density of 1 .5 million cells per mL. 20 mL of the cell suspension were added to the vessels of the invention (also referred to as “baffled tubes”), that were rotated for 24 hours, at 120 rpm for 250 ms clockwise then 120 rpm for 250 ms counterclockwise; collected spheroids were 35-50 pm in diameter.

[0278] Particle (VLP and lentiviruses) production in the vessel of the invention

[0279] All vessels were custom made by 3D printing (capacity : 20 to 80 mL).

[0280] The volume loaded inside the vessel was 20 mL.

[0281] In the performed experiments, the rotation speed was set at 0, 60, 150, 180 rpm, 300, 400, 600, 800, 1200, or 1600 rpm; and the frequency of reversal of rotational direction was set at 0.05 Hz, 0.1 Hz, 0.2 Hz or 2 Hz.

[0282] Cells or spheroids resuspended in an appropriate volume were placed in the vessel, and the vessel was sealed with a vented cap. The complete system (the sealed vessel coupled to a rotating motor) was placed inside the incubator at 37°C for the experiments.

[0283] Preparation of comparative examples, using an orbital shakerand a spinner flask A vessel having a configuration shown in Fig. 12a and Fig. 12b (capacity of 40 mL) was manufactured, using a 3D-printed casting mold from polydimethylsiloxane (Sylgard™). This vessel was placed on an orbital shaker for agitation (hereafter referred to as “orbital shaker”).

[0284] A conventional stirred tank (spinner flask with a capacity of 100 mL) was also used. It consists of a standard spinner flask (supplier Optimus Instruments, CLS-1425-22) and a magnetic stirrer (supplier Avantor, micro-stir 4-pos 391 - 0058). This setup is similar to spinner flask-based systems for extracellular vesicle (EV) production.

[0285] For comparative examples, spheroids were formed in the vessel of the invention in the same way as described above in the section “Small spheroid formation in a vessel of the invention (Examples 6 to 9)”.

[0286] For the particle (VLP and lentivirus) production, the loaded volume was 40mL for the orbital shaker and 60 mL for the spinner flask.

[0287] The rotation speed of the orbital shaker (the speed of the orbital movement) was set at 100 rpm (in other words, 100 orbital movements per minute).

[0288] The rotation speed of the spinner flask (the rotational speed of the stirrer or impeller inside the flask) was set at 100 rpm. Lentiviral titration by the limiting dilution method

[0289] Naive HEK 293T cells were plated the day before lentivirus collection into a 384-well plate at concentration of 7000 cells per well. After lentivirus production, serial dilutions of the collected supernatant from the vessel were prepared, resulting in three final dilutions of 1 :250, 1 :1000, 1 :4000 in complete media, which were then used to incubate the naive plated cells. These cells were incubated with the dilutions for 72h. The 384-well plate was then imaged with a plate-reader with bright-field and with fluorescent microscopy (488 nm). The proportion of fluorescent cells in each well was then assessed as well as the average fluorescence intensity of the cells in the well.

[0290] Example 1 : VLP production in a vessel having a baffle structure comprising solid baffle plates from single (individualized) cells

[0291] Vessels having the configuration shown in Fig. 3a and 3b (vessels having a baffle structure comprising solid baffle plates) were manufactured by 3D printing.

[0292] The experimental conditions were as follows:

[0293] - Producer cells: Human embryonic kidney 293T cells (HEK293T)

[0294] - Capacity of the vessel: 60 mL

[0295] - Cell concentration: 1 million cells per mL

[0296] - Rotation duration (step b)): 2 hours

[0297] - Rotation parameters: rotation speed of 0, 600 and 1200 rpm; rotation freguency of 0.2 Hz

[0298] Prior to the virus-like particle (VLP) production, HEK293T cells were transfected with two plasmids, pVSVG and pWxId, using a conventional transfection protocol. The plasmids pVSVG and pWxId encoded, respectively, a surface vesicular stomatitis virus G (VSVG) protein (which is a fusion protein) and a green fluorescent protein (GFP) (which is used as a non-viral component).

[0299] The transfected cells were imaged by bright-field and fluorescence microscopy (488 nm). The GFP fluorescence of the cells was observed (images not shown), which confirmed that the transfection was successful.

[0300] The transfected HEK293T cells were then, in the form of individualized cells, subjected to rotation under the above conditions.

[0301] The conditioned medium (CM) was analyzed to measure the fluorescence intensity and the number of produced VLPs.

[0302] The results are shown in Fig. 4a (fluorescence intensity) and Fig. 4b (number of produced VLPs). Fig. 4a confirms that GFP signals were detected in the CM after the VLP production, indicating the efficacy of GFP release in the produced VLPs.

[0303] Fig. 4b shows that VLPs were indeed produced after the rotating stimulation, and the number of produced VLPs increased as the rotational speed increased.

[0304] These results demonstrate that the method of the invention is effective for producing protein-loaded biological particles from individualized producer cells.

[0305] Example 2a: VLP production in a vessel having a baffle structure comprising solid baffle plates from spheroids formed in a microwell

[0306] The same vessels were used as in Example 1 .

[0307] The experimental conditions were as follows:

[0308] - Producer cells: spheroids made of HEK293T cells

[0309] - Spheroid concentration: 2000-4000 per mL

[0310] - Rotation duration (step (b)): 2 hours

[0311] - Rotation parameters: rotation speed of 0 and 600 rpm and rotation freguency of 0.2 Hz

[0312] Prior to the VLP production, HEK293T spheroids were prepared in agarose microwells, according to the conventional protocol (average spheroid diameter: 100 to 150 pm).

[0313] HEK293T cells were transfected, using the conventional transfection protocol, with the same plasmids as in Example 1 , either before the spheroid formation or after the spheroid formation. In the latter case, i.e., transfection of spheroids, the spheroid fusion resulted in larger spheroids, in a diameter range of 250-500 pm.

[0314] The transfected HEK293T spheroids were imaged by bright-field and fluorescence microscopy (488 nm). The GFP fluorescence of the spheroids was observed (images not shown), which confirmed that the transfection was successful.

[0315] The spheroids were then subjected to rotation under the above conditions, and the CM was analyzed to measure the fluorescence intensity and the number of produced VLPs.

[0316] The results are shown in Fig. 5a (fluorescence intensity) and Fig. 5b (number of produced VLPs). In both cases of transfection after spheroid formation (1) and before spheroid formation (2), Fig. 5a confirms that GFP signals were detected in the CM, indicating the efficacy of GFP release in the produced VLPs; additionally, Fig. 5b shows that VLPs were indeed produced after the rotating stimulation. These results demonstrate that the method of the invention is effective for producing protein-loaded biological particles from producer cells in the form of spheroids.

[0317] Example 2b: VLP production in a vessel having a baffle structure comprising solid baffle plates from spheroids formed in the vessel

[0318] The same vessels were used as in Example 1 .

[0319] The experimental conditions were as follows.

[0320] - Producer cells: spheroids made of HEK293T cells

[0321] - Spheroid concentration: 2000-4000 per mL

[0322] - Rotation parameters for spheroid maturation: 48 hours at 60 rpm

[0323] - Rotation parameters for VLP production: 2 hours at 0 to 1200 rpm and at 0.2 Hz

[0324] Prior to the VLP production, HEK293T cells were transfected, using the conventional transfection protocol, with the same plasmids as in Example 1 .

[0325] The transfected cells in suspension were directly supplied in the vessel and spheroids were formed, as described above in the section “Spheroid formation in a vessel of the invention (Examples 2 to 5)”.

[0326] The transfected HEK293T spheroids were imaged by bright-field and fluorescence microscopy (488 nm). The GFP fluorescence of the spheroids was observed (images not shown), which confirmed that the transfection was successful.

[0327] The spheroids were then subjected to rotation in the vessel under the conditions above to trigger the production of VLPs.

[0328] The CM was analyzed to measure the fluorescence intensity and the number of produced VLPs. The results are shown in Fig. 6a (fluorescence intensity) and Fig. 6b (number of produced VLPs).

[0329] Fig. 6a and Fig. 6b show similar results as in Fig. 5, indicating the efficacy of GFP release in the produced VLPs (Fig. 6a) and VLP production by the rotating stimulation (Fig. 6b).

[0330] These results also demonstrate that the relative fluorescence units (RFU) and the number of produced VLPs increase as the rotational speed increased, with the rotational speed of 600 rpm an optimal rotational speed; and that the method of the invention is effective for streamlining the formation of spheroids and producing protein-loaded biological particles from such spheroid producer cells.

[0331] The VLPs produced from spheroids formed in microwells as in Example 2a (transfection before spheroid formation, and rotation at 600 rpm) and formed in the vessel of the invention as in this Example 2b (rotation at 600 rpm) were analyzed by NanoFCM for the GFP fluorescence of single particles.

[0332] The results are shown in Fig. 7, confirming that each produced particle was indeed associated with a GFP fluorescent signal (FITC+).

[0333] Example 3: Lentivirus production in a vessel having a baffle structure comprising solid baffle plates from spheroids formed in the vessel

[0334] The same vessels were used as in Example 1 .

[0335] The experimental conditions were as follows.

[0336] - Producer cells: HEK293T spheroids

[0337] - Spheroid diameter: 120 pm

[0338] - Spheroid concentration: 1000-2000 per mL

[0339] - Rotation parameters during spheroid maturation: 12 hours at 60 rpm

[0340] - Rotation parameters during lentivirus production: o Duration: 36 hours, consisting of 9 cycles; each cycle consists of 40 minutes of rapid stimulation and 3 hours and 20 minutes of slow stimulation o Rotational speed: 150, 300, 600 or 1200 rpm for rapid stimulation and 60 rpm for slow stimulation o Acceleration rates during the rapid stimulation: high acceleration (600 rpm at 0.2 Hz) and low acceleration (100-300 rpm at 0.1 Hz)

[0341] Prior to the lentivirus production, HEK293T cells were transfected with a plasmid psPAX2 in addition to pVSVG and pWxId plasmids, using a conventional transfection protocol. The plasmid psPAX2 encodes genes necessary for the assembly and formation of the capsid which facilitates RNA export.

[0342] The transfected cells were directly placed in the vessel under the above spheroid maturation conditions.

[0343] The transfected spheroids were imaged by bright-field and fluorescence microscopy (488 nm). The GFP fluorescence of the spheroids was observed (images not shown), confirming that the transfection was successful.

[0344] The CM was analyzed to measure the fluorescence intensity. The results are shown in Fig. 8

[0345] Fig. 8 shows that GFP signals were detected in the CM after the lentivirus production, indicating the efficacy of GFP release in the produced lentiviruses.

[0346] Thus, the results demonstrate that the method of the invention is effective for producing lentiviruses from producer cells in the form of spheroids, and that that production can be enhanced via stimulation parameters. It is also to be noted that the optimal rotation speeds are lower than those required to trigger the production of VLPs.

[0347] Example 4: Transduction of HeLa cells with the lentiviruses produced according to the invention

[0348] HeLa cells were transduced with serial dilutions of lentiviruses produced according to the invention, using a conventional transduction protocol.

[0349] The expression of GFP was measured by flow cytometry 4 days after the transduction, and represented as percentage of cells expressing GFP (Fig. 9a) and mean fluorescence intensity (MFI) (Fig. 9b).

[0350] As shown in Fig.9a, at a 1 / 2 dilution, the percentage of transduced cells expression GFP was similar across all tested conditions, approximately 100%. At lower dilutions, the percentage of GFP-expressing cells decreased but remained higher than those transduced using the traditional method for lentivirus production (2D) (CTL), except for spheroids stimulated at 0 rpm (0).

[0351] Fig. 9b demonstrates that the MFI was higher than those transduced using the traditional method (CTL), except for spheroids stimulated at 0 rpm (0). The best results were observed for the lentiviruses produced from spheroids stimulated at 60 / 300 rpm (3), 60 / 600 rpm (4) and 150 rpm (5).

[0352] The expression of GFP was also measured by flow cytometry 7 days after the transduction (Fig. 10a and Fig. 10b). The results were similar to those shown in Fig. 9, indicating that the GFP expression remained stable in the cells 7 days after the transduction.

[0353] These results show that the use of lentiviruses produced according to the method of the invention improves the transduction efficiency of Hela cells. This is most probably due to the increase of the amount of biological particles produced using the method of the invention.

[0354] Example 5: Transduction of Jurkat cells with the lentiviruses produced according to the invention

[0355] Jurkat cells were transduced with serial dilutions of lentiviruses produced according to the invention, using a conventional transduction protocol.

[0356] The expression of GFP was measured by flow cytometry 7 days after the transduction, and represented as percentage of cells expressing GFP (Fig. 11a) and mean fluorescence intensity (MFI) (Fig. 11b).

[0357] As shown in Fig. 11a, the percentage of transduced cells expression GFP was higher than those transduced using the traditional method for lentivirus production (2D) (CTL), except for spheroids stimulated at 0 rpm (0). In particular for the MFI (Fig. 11b), good results were obtained for the spheroids stimulated at 60 / 150 rpm (2), 60 / 300 rpm (3), 150 rpm (5) and 300 rpm (6).

[0358] These results show that the use of lentiviruses produced according to the method of the invention improves the transduction efficiency of Jurkat cells. This is most probably due to the increase of the amount of biological particles produced using the method of the invention.

[0359] Example 6: VLP production using the vessel of the invention and an orbital shaker from producer cells transfected with a plasmid encoding a fusion protein

[0360] VLP production using the method of the invention was compared with VLP production using an orbital shaker.

[0361] Spheroids made of HEK293T cells were used as producer cells.

[0362] For the vessel of the invention, the same vessels were used as in Example 1 .

[0363] A baffled orbital shaker was prepared as described above in the section “Preparation of comparative examples, using an orbital shaker and a spinner flask".

[0364] The rotation parameters for VLP production were as follows.

[0365] - Vessel of the invention: 600 rpm at 0.2 Hz for 3 hours

[0366] - Orbital shaker: orbital rotation of 100 rpm for 3 hours

[0367] It is to be noted that the speed of 100 rpm for the orbital shaker is a common attainable rotational speed in a conventional orbital shaker.

[0368] HEK293T spheroids were formed from cells in suspension, as described above. The spheroids were further incubated for 48h in the vessel of the invention.

[0369] Prior to the VLP production, the spheroids were transfected with two plasmids, pVSVG and pWxId in the vessel of the invention, using a conventional transfection protocol for 18h.

[0370] The transfected spheroids were imaged by bright-field and fluorescence microscopy (488 nm). The GFP fluorescence of the spheroids was observed (images not shown), which confirmed that the transfection was successful.

[0371] The transfected spheroids were transferred into new vessels (the vessel of the invention or the orbital shaker) and subjected to rotation under the above conditions to trigger the production of VLPs.

[0372] The CM was analyzed by measuring the particle yield (the number of produced VLPs per cell introduced at the beginning of the experiment) with Videodrop and the fluorescence intensity with a plate reader. The VLPs in the CM were then concentrated. This concentrated CM was also analyzed for the fluorescence intensity to better distinguish the particle- associated fluorescence from the background signal.

[0373] The results are shown in Fig. 13a (the number of produced VLPs per cell) and Fig. 13b (the fluorescence intensity), including the results of a negative control (NC, 0 rpm in a non-adherent cell culture flask for 3h). For Fig. 13b, the results of a baseline control (TO, initial state at time 0) are also included.

[0374] These results show that the method of the invention (A) resulted in increased VLP production compared to the method using an orbital shaker (B), indicating that it favors the formation of VLP particles and is more efficient than the baffled orbital shaker (Fig. 13a).

[0375] This increased production corelates with higher fluorescence level, especially after the concentration of produced VLPs (Fig. 13b): while concentrating the VLP particles did not increase the fluorescence in the condition with the orbital shaker (B), it resulted in more than a 4-fold increase in fluorescence in the condition with the vessel of the invention (A) (light-gray bar on the right hand-side). This demonstrates that the vessel of the invention is more efficient in generation of protein-loaded biological particles.

[0376] Example 7: VLP production using the vessel of the invention and an orbital shaker from producer cells transfected with a plasmid encoding a capsid protein

[0377] The experimental conditions and procedures were the same as in Example 6, except that HEK293T cells were transfected with a psPAX2 plasmid (encoding a capsid protein) and pWxId.

[0378] As a control for the effect of psPAX2 plasmid, half of the spheroids were processed in the same way but without any plasmids.

[0379] The successful transfection was confirmed in the same way as in Example 6.

[0380] The transfected and non-transfected spheroids were subjected to rotation; the CM was analyzed; and the produced VLPs in the CM were concentrated in the same way as in Example 6.

[0381] This concentrated CM was then processed through an RNA isolation kit (RNA Nucluospin, Macherey-Nagel) and the amount of RNA loaded into the produced VLPs were measured with a Nanodrop.

[0382] The results are shown in Fig. 14a (the number of produced VLPs per cell), Fig. 14b (the fluorescence intensity), and Fig. 14c (the nucleic acid concentration in the CM), including the results of a negative control (NC, 0 rpm in a non-adherent cell culture flask for 3h). For Fig. 14b, the results of a baseline control (TO, initial state at time 0) are also included.

[0383] As in Example 6, the method of the invention (A-2) resulted in an increased production of VLPs compared to the method using an orbital shaker (B-2) (Fig. 14a).

[0384] In addition, only the CM from the method of the invention (A-2) showed fluorescence levels above the baseline control (TO-2) (Fig. 14b), indicating that only the method of the invention led to the production of protein-loaded particles from the transfected spheroids.

[0385] Fig. 14c further shows that only the particles produced in the method of the invention (A-2) were loaded with a significant amount of RNA (Fig. 14c). This indicates that the method of the invention improves the efficiency of production of RNA-loaded biological particles mediated by a capsid protein, such as GAG protein.

[0386] Example 8: Lentivirus production using the vessel of the invention, an orbital shaker and a spinner flask

[0387] Lentivirus production using the method of the invention was compared with lentivirus production using an orbital shaker and a conventional spinner flask.

[0388] Spheroids made of HEK293T cells were used as producer cells.

[0389] For the vessel of the invention, the same vessels were used as in Example 1 . A baffled orbital shaker and a spinner flask were prepared as described above in the section “Preparation of comparative examples, using an orbital shakerand a spinner flask

[0390] The rotation parameters for lentivirus production were as follows.

[0391] - Vessel of the invention: 600 rpm at 0.1 Hz for 48 hours

[0392] - Orbital shaker: orbital rotation of 100 rpm for 48 hours

[0393] - Spinner flask: 100 rpm rotation for 48 hours

[0394] It is to be noted that the speed of 100 rpm for the orbital shaker and the spinner flask is a common attainable rotational speed in a conventional orbital shaker and in a conventional spinner flask.

[0395] HEK293T spheroids were formed from cells in suspension as described above in the sections “Spheroid formation in a vessel of the invention (Examples 6 to 9)” and “Preparation of comparative examples". Prior to lentivirus production, the spheroids were transfected with two plasmids, pVSVG and psPAX2 (thus allowing complete lentiviruses to be formed) in addition to the pWxId plasmid, in the vessel of the invention, using a conventional transfection protocol for 18h. The successful transfection was confirmed in the same way as in Example 6.

[0396] The transfected spheroids were transferred into new vessels (the vessel of the invention, the orbital shaker, or the spinner flask) and subjected to rotation under the above conditions to trigger the production of lentiviruses.

[0397] Lentiviruses were collected after 48h of production, and titrated as described above in the section “Lentiviral titration by the limiting dilution method’.

[0398] The results are shown in Fig. 15a (the average percentage of the fluorescent cells for each dilution) and in Fig. 15b (the average fluorescence of the cells).

[0399] As shown in Fig. 15a and Fig. 15b, overall, at every dilution, the supernatant obtained by the method of the invention (A) had higher fluorescence levels, indicating a higher percentage of transduced cells and a higher titer of the produced lentiviruses in the supernatant obtained from the vessel of the invention.

[0400] These results demonstrate that the vessel of the invention is more efficient in the lentivirus production than a baffled orbital shaker or a spinner flask.

[0401] Example 9: Effect of repeatedly changing the rotational motion of the vessel on the lentivirus production

[0402] Lentivirus production was performed using the method of the invention, with variations in rotational motion parameters, and also using an orbital shaker.

[0403] The experimental conditions and procedures were the same as in Example 8, except that the spinner flask was not used and that the rotation parameters for lentivirus production were as follows:

[0404] - Vessel of the invention o a constant rotation speed of 600 rpm (0 Hz) (not in accordance with the method of the invention); o a fast-changing rotation of 180 rpm at 2 Hz; o a rotation of 600 rpm at 0.1 Hz; o a rotation of 600 rpm at 0.2 Hz during 30 min followed by a rest time of 30 min

[0405] - Orbital shaker: an orbital rotation of 100 rpm.

[0406] The transfected spheroid formation, lentivirus production and the titration of the lentiviruses were performed in the same way as in Example 8.

[0407] The results are shown in Fig. 16a (the average percentage of the fluorescent cells for each dilution) and in Fig. 16b (the average fluorescence of the cells). As shown in these figures, the supernatant obtained under the rotation of 600 rpm at 0 Hz (NC) had a lower percentage of transduced cells and lower fluorescence levels of the cells than all the other rotation conditions in accordance with the invention (A1 , A2 and A3). Notably, the rotation of 600 rpm at 0 Hz (NC) resulted in a lower percentage of transduced cells and lower fluorescence levels of the cells than the rotation condition that differed by the presence of the directional change at 0.1 Hz (A2).

[0408] These results demonstrate the importance of the direction change of the rotations in the invention to generate a sufficient amount of shear stress to trigger more lentivirus production.

[0409] In addition, for the vessel of the invention, overall, the rotation of 180 rpm at 2 Hz (A1) led to the highest titer, followed by the rotation of 600 rpm at 0.1 Hz (A2) and then finally the rotation of 600 rpm at 0.2 Hz with rest times (A3). This demonstrates that a more frequent change of direction with an optimal rotation speed produces a stress that is more favorable for lentivirus production. Indeed, increasing the shear stress by increasing the rotation speed does not necessarily lead to an optimal lentivirus yield if the rotational motions are not repeatedly changed.

[0410] Regarding the orbital rotation of a vessel, the supernatant obtained from the orbital shaker (B) had an even lower percentage of transduced cells lower fluorescence levels of the cells than the negative control (NC), indicating that the orbital shaking, although producing periodic changes in the flow, is not efficient in generation of the appropriate hydrodynamic stresses.

Claims

CLAIMS1. A method of producing biological particles comprising a viral or virus-like component from producer cells, comprising the steps of: a) placing producer cells in a liquid medium in a vessel comprising a baffle structure fixed inside the vessel; b) rotating the vessel so as to generate biological particles comprising a viral or virus-like component from the producer cells; and c) collecting the generated biological particles comprising a viral or virus-like component; wherein the step b) of rotating the vessel comprises repeatedly changing the rotational motion of the vessel.

2. The method of claim 1 , wherein the viral or virus-like component comprises a viral protein and / or a nucleic acid encoding the viral protein, the viral protein preferably comprising a structural protein such as a fusion protein, an envelope protein, a capsid protein, and other proteins involved in the virus structure, or a combination thereof.

3. The method of claim 1 or 2, wherein the biological particles are selected from viruses or virus-like particles.

4. The method of any one of claims 1 to 3, wherein the viruses are selected from retroviruses such as lentiviruses and Gamma-Retrovirus; adenoviruses; and Baboon virus, preferably lentiviruses.

5. The method of any one of claims 1 to 4, wherein the biological particles are non-pathogenic, more preferably replication-deficient.

6. The method of any one of claims 1 to 5, wherein the biological particles further comprises a non-viral molecule, preferably a therapeutic molecule, a biomarker, a bioactive protein or nucleic acid, an aptamer, an antibody and / or a molecular target.

7. The method of any one of claim 6, further comprising, prior to step a), a step of introducing the non-viral molecule into the producer cells.

8. The method of any one of claims 1 to 7, further comprising, prior to step a), a step of introducing at least one nucleic acid encoding the viral or viruslike component into the producer cells.

9. The method of any one of claims 1 to 8, wherein the vessel rotates around a rotation axis which is substantially vertically oriented.

10. The method of any one of claims 1 to 9, wherein the biological particles are generated from producer cells in the form of spheroids.

11. The method of claim 10, wherein:- the method further comprises a preliminary step of growing spheroids outside of the vessel, and step a) of placing the producer cells in the vessel comprises supplying the grown spheroids to the vessel; or- step a) of placing the producer cells in the vessel comprises supplying individualized cells to the vessel, and the method further comprising an intermediate step of generating spheroids from the individualized cells.

12. The method of any one of claims 1 to 11 , wherein the step b) of rotating the vessel comprises periodically changing the rotational motion of the vessel, preferably step b) comprises repeatedly reversing the rotational direction of the vessel, repeatedly changing the rotational speed of the vessel, and / or intermittently rotating the vessel.

13. The method of claim 12, wherein the frequency of changing the rotational motion of the vessel is from 0.01 to 5 Hz, preferably from 0.05 to 0.5 Hz.

14. The method of any one of claims 1 to 13, wherein the step b) of rotating the vessel is carried out at a maximum rotational speed of 50 to 6000 rpm, preferably 150 to 1200 rpm.

15. The method of any one of claims 1 to 14, further comprising repeating cycles of at least steps b) and c), using the same producer cells.

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